Heterocyclic compounds for the inhibition of pask

ABSTRACT

Disclosed herein are new heterocyclic compounds and compositions and their application as pharmaceuticals for the treatment of disease. Methods of inhibiting PAS Kinase (PASK) activity in a human or animal subject are also provided for the treatment of diseases such as diabetes mellitus.

This application is a continuation of U.S. application Ser. No.14/438,268, filed Apr. 24, 2015, which claims the benefit of priorityunder 35 U.S.C. §371 of PCT International Application No.PCT/US2013/066782, filed Oct. 25, 2013, which in turn claims the benefitof priority of U.S. Provisional Application No. 61/718,487, filed Oct.25, 2012, the disclosures of which are hereby incorporated by referenceas if written herein in their entireties.

Disclosed herein are new heterocyclic compounds and compositions andtheir application as pharmaceuticals for the treatment of disease.Methods of inhibiting PAS Kinase (PASK) activity in a human or animalsubject are also provided for the treatment of diseases such as diabetesmellitus.

The regulation of glycogen metabolism is critical for the maintenance ofglucose and energy homeostasis in mammals. Glycogen, a large branchedpolymer of glucose, acts as a reserve of carbon and energy in a varietyof organisms. In mammals, the most important stores are found in theliver and skeletal muscle (1). Liver glycogen is required to efficientlybuffer blood glucose levels during fasting, whereas muscle glycogen isprimarily used locally as a fuel for muscle contraction (2).Dysregulation of glycogen metabolism has been implicated in thedevelopment of many diseases, including type 2 diabetes mellitus (3, 4).

The synthesis of glycogen is primarily controlled through regulation ofthe enzyme glycogen synthase (GYS, various isoforms), which catalyzesbulk glycogen synthesis (5, 6, 7). The muscle isoform of glycogensynthase (GYS1) is inactivated by reversible phosphorylation that occursat nine distinct sites within the enzyme (8, 9, 10). In the bestcharacterized form of glycogen synthase, the phosphorylation sites areclustered at the N and C termini (14). Glycogen synthase kinase-3(GSK-3), an insulin-dependent kinase which has long been implicated inthe stepwise phosphorylation of four key sites in the C terminus ofglycogen synthase including Ser-640 (one of the most importantendogenous regulatory phosphorylation sites in mammalian glycogensynthase (15, 32) and Ser-644 (10, 11-13, 24, 25). GSK-3, however, isnot the sole kinase that phosphorylates C-terminal regulatory sites;GSK-3-independent mechanisms also exist, since serine-to-alaninesubstitutions at Ser-7 and Ser-10 block GSK-3-mediated phosphorylationof the important regulatory sites Ser-640 and Ser-644, andphosphorylation at these sites still occurs.

PASK (purine-analog sensitive kinase, PAS kinase) is a PASdomain-containing serine/threonine kinase, and genetic experiments in S.cerevisiae yeast have implicated PASK as a physiological regulator ofglycogen synthase and glycogen accumulation (16, 17). As with the entireglycogen synthase regulatory system, PASK is highly conserved from yeastto man. Human PASK (hPASK) phosphorylates glycogen synthase primarily atSer-640, causing near complete inactivation. It is interesting to notethat the exact site of PASK-dependent phosphorylation is similar but notidentical in yeast and mammalian glycogen synthase (18, 19); yeast PASKphosphorylates glycogen synthase at the site analogous to Ser-644, fourresidues C-terminal (18). It appears that the hPASK mid region (residues444-955) is required for efficient phosphorylation of glycogen synthasein vitro and for interaction with glycogen synthase in cells: an hPASKmutant (Δ955) lacking the noncatalytic N terminus was unable toefficiently phosphorylate glycogen synthase. Since this region is notrequired for the phosphorylation of generic, nonphysiologicalsubstrates, such as histones and synthetic peptides, it has beenproposed that the mid region of hPASK is essential forsubstrate-targeting. A similar substrate region has been discovered inmany protein kinases (26-29). Unlike GSK-3, the activity of hPASK hasbeen shown to be independent of insulin and probably regulated insteadby a more direct metabolic signal (23).

Genetic and proteomic screens using yeast PASK identified a number ofsubstrates and implicated this kinase in the regulation of carbohydratemetabolism and translation (18). It has previously been shown that yeastPASK phosphorylates glycogen synthase in vitro and that strains lackingthe PASK genes (PSK1 and PSK2) had elevated glycogen synthase activityand an approximately 5- to 10-fold accumulation of glycogen relative towild-type strains, consistent with impaired ability to phosphorylateglycogen synthase in vivo (18). Because glycogen synthesis andtranslation are two processes tightly regulated in response to nutrientavailability and because PAS domains are frequently involved inmetabolic sensing, a role for PASK in the cellular response to metabolicstatus has been proposed. Indeed, it was recently demonstrated thatmammalian PASK plays a role in the cellular response to nutrients. Thecatalytic activity of PASK in pancreatic islet β-cells is rapidlyincreased in response to glucose addition, and PASK is required for theglucose-responsive expression of some β-cell genes, includingpreproinsulin (23).

PASK catalytic activity is not responsive to glucose alone, however. Theinteraction between the hPASK midregion and glycogen synthase isregulated by at least two factors. First, the PAS domain of PAS kinaseplays a negative role in regulating this interaction. If the PAS domainis deleted or disrupted, hPASK associates more stably with glycogensynthase. PAS domain function is usually controlled by the metabolicstatus of the host cell, as has been suggested for the PASK PAS domain(23). This observation raises the intriguing possibility that thehPASK-glycogen synthase interaction is regulated by the metabolic statusof the cell, thereby enabling an additional layer of metabolicregulation of glycogen synthesis. Second, glycogen negatively regulatesthe hPASK-glycogen synthase interaction, which would initially seemcounterintuitive, since glycogen would thereby stimulate its owncontinued synthesis. It is possible, however, that this mechanism existsto spatially coordinate the synthesis of glycogen. It is becomingincreasingly apparent that glycogen is synthesized in cells in a highlyorganized spatial pattern (30). Perhaps one function of hPASK is tomaintain free, unlocalized glycogen synthase in a phosphorylated,inactive form until it is properly localized to an existing, properlyorganized glycogen particle. These data strongly suggest that the hPASKmidregion plays an important role in targeting hPASK catalytic activityto specific substrates within the cell.

Since hPASK has been recently implicated in glucose-sensing andglucose-responsive transcription, it appears likely that glucosesignaling by means of hPASK affects glycogen metabolism in vivo. It iswell-established that derangement in glycogen metabolism is one of thehallmarks of both Type 1 and Type 2 diabetes (20) and related conditions(21), including a panoply of life-threatening cardiovascular conditions(22). Using PASK1 mice, it has further been demonstrated that PASK isindeed required for normal insulin secretion by pancreatic β cells, andthat PASK deletion results in nearly complete resistance to thephenotypes caused by a high-fat diet, including obesity, insulinresistance and hepatic fat accumulation. Therefore, PASK inhibitionwould comprise a system for the metabolic control of glucose utilizationand storage in mammalian cells, and offer a new method to treatmetabolic diseases including but not limited to diabetes and itscomplications, the metabolic syndrome, insulin resistance, and variouscardiovascular conditions.

Novel compounds and pharmaceutical compositions, certain of which havebeen found to inhibit PASK have been discovered, together with methodsof synthesizing and using the compounds including methods for thetreatment of PASK-mediated diseases in a patient by administering thecompounds.

In an embodiment, compounds have structural Formula I

-   -   or a pharmaceutically acceptable salt, ester, or prodrug        thereof, wherein:    -   R₁ is chosen from hydrogen, aryl, alkyl, arylalkyl, heteroaryl,        heteroarylalkyl, and CONR₁₀R₁₁, any of which may be optionally        substituted.    -   R₂ is chosen from aryl and heteroaryl, either of which may be        optionally substituted; and    -   R₁₀ and R₁₁ are each independently chosen from hydrogen, lower        alkyl, aryl, or optionally, R₁₀ and R₁₁ can be taken together to        form a heterocycloalkyl, or heteroaryl.

Certain compounds disclosed herein may possess useful PASK modulatingactivity, and may be used in the treatment or prophylaxis of a diseaseor condition in which PASK plays an active role. Thus, in broad aspect,certain embodiments also provide pharmaceutical compositions comprisingone or more compounds disclosed herein together with a pharmaceuticallyacceptable carrier, as well as methods of making and using the compoundsand compositions. Certain embodiments provide methods for modulatingPASK. Other embodiments provide methods for treating a PASK-mediateddisorder in a patient in need of such treatment, comprisingadministering to said patient a therapeutically effective amount of acompound or composition according to the present invention. Alsoprovided is the use of certain compounds disclosed herein for use in themanufacture of a medicament for the treatment of a disease or conditionameliorated by the inhibition of PASK.

Further provided is a compound as disclosed herein together with apharmaceutically acceptable carrier

Further provided is a compound as disclosed herein for use as amedicament.

Further provided is a compound as disclosed herein for use in themanufacture of a medicament for the prevention or treatment of a diseaseor condition ameliorated by the inhibition of PASK.

Further provided is a compound as disclosed herein for use in themanufacture of a medicament for the prevention or treatment of a diseaseor condition ameliorated by the inhibition of PASK.

Provided herein is a compound as disclosed herein for use in theprevention or treatment of a disease or condition ameliorated by theinhibition of PASK.

Further provided is a pharmaceutical composition comprising a compoundas recited above together with a pharmaceutically acceptable carrier.

Further provided is a method of inhibiting PASK comprising contactingPASK with a compound as disclosed herein.

Further provided is a method of treatment of a disease comprising theadministration of a therapeutically effective amount of a compound asdisclosed herein to a patient in need thereof.

Further provided is the method as recited above wherein said disease ischosen from cancer and a metabolic disease.

Further provided is the method as recited above wherein said disease isa metabolic disease.

Further provided is the method as recited above wherein said metabolicdisease is chosen from metabolic syndrome, diabetes, dyslipidemia, fattyliver disease, non-alcoholic steatohepatitis, obesity, and insulinresistance.

Further provided is the method disclosed herein wherein said diabetes isType II diabetes.

Further provided is the method as disclosed herein wherein saiddyslipidemia is hyperlipidemia.

Further provided is the method wherein said hyperlipidemia ishypertriglyceridemia.

Further provided is a method for achieving an effect in a patientcomprising the administration of a therapeutically effective amount of acompound as disclosed herein to a patient, wherein the effect isselected from the group consisting of reduction of triglycerides,reduction of cholesterol, and reduction of hemoglobin A1c.

Further provided is the method as disclosed herein wherein saidcholesterol is chosen from LDL and VLDL cholesterol.

Further provided is the method as disclosed herein wherein saidtriglycerides are chosen from plasma triglycerides and livertriglycerides.

Further provided is a method of treatment of a PASK-mediated diseasecomprising the administration of:

-   -   a. a therapeutically effective amount of a compound as disclosed        herein; and    -   b. another therapeutic agent.

Not to be bound by any theory or mechanism, the compounds disclosedherein can be used to treat or modulate metabolic disease (including butnot limited to diabetes, metabolic disorder, dyslipidemia, fatty liverdisease, non-alcoholic steatohepatitis, obesity, and insulin resistance,as well as to reduce triglycerides, cholesterol, and hemoglobin A1c) andcancer.

As used herein, the terms below have the meanings indicated.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” is used, where n₁ and n₂ are the numbers, then unless otherwisespecified, this notation is intended to include the numbers themselvesand the range between them. This range may be integral or continuousbetween and including the end values. By way of example, the range “from2 to 6 carbons” is intended to include two, three, four, five, and sixcarbons, since carbons come in integer units. Compare, by way ofexample, the range “from 1 to 3 μM (micromolar),” which is intended toinclude 1 μM, 3 μM, and everything in between to any number ofsignificant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “acyl,” as used herein, alone or in combination, refers to acarbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl,heterocycle, or any other moiety were the atom attached to the carbonylis carbon. An “acetyl” group refers to a —C(O)CH₃ group. An“alkylcarbonyl” or “alkanoyl” group refers to an alkyl group attached tothe parent molecular moiety through a carbonyl group. Examples of suchgroups include methylcarbonyl and ethylcarbonyl. Examples of acyl groupsinclude formyl, alkanoyl and aroyl.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkenyl will comprise from 2 to 6 carbon atoms. Theterm “alkenylene” refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene [(—CH═CH—), (—C:C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like. Unless otherwisespecified, the term “alkenyl” may include “alkenylene” groups.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 20carbon atoms. In certain embodiments, said alkyl will comprise from 1 to10 carbon atoms. In further embodiments, said alkyl will comprise from 1to 6 carbon atoms. Alkyl groups may be optionally substituted as definedherein. Examples of alkyl radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl,hexyl, octyl, noyl and the like. The term “alkylene,” as used herein,alone or in combination, refers to a saturated aliphatic group derivedfrom a straight or branched chain saturated hydrocarbon attached at twoor more positions, such as methylene (—CH₂—). Unless otherwisespecified, the term “alkyl” may include “alkylene” groups.

The term “alkylamino,” as used herein, alone or in combination, refersto an alkyl group attached to the parent molecular moiety through anamino group. Suitable alkylamino groups may be mono- or dialkylated,forming groups such as, for example, N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

The term “alkylidene,” as used herein, alone or in combination, refersto an alkenyl group in which one carbon atom of the carbon-carbon doublebond belongs to the moiety to which the alkenyl group is attached.

The term “alkylthio,” as used herein, alone or in combination, refers toan alkyl thioether (R—S—) radical wherein the term alkyl is as definedabove and wherein the sulfur may be singly or doubly oxidized. Examplesof suitable alkyl thioether radicals include methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio,tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkynyl comprises from 2 to 6 carbon atoms. In furtherembodiments, said alkynyl comprises from 2 to 4 carbon atoms. The term“alkynylene” refers to a carbon-carbon triple bond attached at twopositions such as ethynylene (—C:C—, —C≡C—). Examples of alkynylradicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl,butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like.Unless otherwise specified, the term “alkynyl” may include “alkynylene”groups.

The terms “amido” and “carbamoyl,” as used herein, alone or incombination, refer to an amino group as described below attached to theparent molecular moiety through a carbonyl group, or vice versa. Theterm “C-amido” as used herein, alone or in combination, refers to a—C(═O)—NR₂ group with R as defined herein. The term “N-amido” as usedherein, alone or in combination, refers to a RC(═O)NH— group, with R asdefined herein. The term “acylamino” as used herein, alone or incombination, embraces an acyl group attached to the parent moietythrough an amino group. An example of an “acylamino” group isacetylamino (CH₃C(O)NH—).

The term “amino,” as used herein, alone or in combination, refers to—NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl,heteroaryl, and heterocycloalkyl, any of which may themselves beoptionally substituted. Additionally, R and R′ may combine to formheterocycloalkyl, either of which may be optionally substituted.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch polycyclic ring systems are fused together. The term “aryl”embraces aromatic groups such as phenyl, naphthyl, anthracenyl, andphenanthryl.

The term “arylalkenyl” or “aralkenyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkenyl group.

The term “arylalkoxy” or “aralkoxy,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkoxy group.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group.

The term “arylalkynyl” or “aralkynyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkynyl group.

The term “arylalkanoyl” or “aralkanoyl” or “aroyl,” as used herein,alone or in combination, refers to an acyl radical derived from anaryl-substituted alkanecarboxylic acid such as benzoyl, napthoyl,phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl,(2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

The term aryloxy as used herein, alone or in combination, refers to anaryl group attached to the parent molecular moiety through an oxy.

The terms “benzo” and “benz,” as used herein, alone or in combination,refer to the divalent radical C⁶H⁴=derived from benzene. Examplesinclude benzothiophene and benzimidazole.

The term “carbamate,” as used herein, alone or in combination, refers toan ester of carbamic acid (—NHCOO—) which may be attached to the parentmolecular moiety from either the nitrogen or acid end, and which may beoptionally substituted as defined herein.

The term “O-carbamyl” as used herein, alone or in combination, refers toa —OC(O)NRR′, group—with R and R′ as defined herein.

The term “N-carbamyl” as used herein, alone or in combination, refers toa ROC(O)NR′— group, with R and R′ as defined herein.

The term “carbonyl,” as used herein, when alone includes formyl [—C(O)H]and in combination is a —C(O)— group.

The term “carboxyl” or “carboxy,” as used herein, refers to —C(O)OH orthe corresponding “carboxylate” anion, such as is in a carboxylic acidsalt. An “O-carboxy” group refers to a RC(O)O— group, where R is asdefined herein. A “C-carboxy” group refers to a —C(O)OR groups where Ris as defined herein.

The term “cyano,” as used herein, alone or in combination, refers to—CN.

The term “cycloalkyl,” or, alternatively, “carbocycle,” as used herein,alone or in combination, refers to a saturated or partially saturatedmonocyclic, bicyclic or tricyclic alkyl group wherein each cyclic moietycontains from 3 to 12 carbon atom ring members and which may optionallybe a benzo fused ring system which is optionally substituted as definedherein. In certain embodiments, said cycloalkyl will comprise from 3 to7 carbon atoms. Examples of such cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronapthyl,indanyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and thelike. “Bicyclic” and “tricyclic” as used herein are intended to includeboth fused ring systems, such as decahydronaphthalene,octahydronaphthalene as well as the multicyclic (multicentered)saturated or partially unsaturated type. The latter type of isomer isexemplified in general by, bicyclo[1,1,1]pentane, camphor, adamantane,and bicyclo[3,2,1]octane.

The term “ester,” as used herein, alone or in combination, refers to acarboxy group bridging two moieties linked at carbon atoms.

The term “ether,” as used herein, alone or in combination, refers to anoxy group bridging two moieties linked at carbon atoms.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “haloalkoxy,” as used herein, alone or in combination, refersto a haloalkyl group attached to the parent molecular moiety through anoxygen atom.

The term “haloalkyl,” as used herein, alone or in combination, refers toan alkyl radical having the meaning as defined above wherein one or morehydrogens are replaced with a halogen. Specifically embraced aremonohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkylradical, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Haloalkylene” refers to a haloalkyl group attached attwo or more positions. Examples include fluoromethylene (—CFH—),difluoromethylene (—CF₂—), chloromethylene (—CHCl—) and the like.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain hydrocarbon radical, orcombinations thereof, fully saturated or containing from 1 to 3 degreesof unsaturation, consisting of the stated number of carbon atoms andfrom one to three heteroatoms selected from the group consisting of O,N, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be substituted orquaternized. The heteroatom(s) O, N and S may be placed at any interiorposition of the heteroalkyl group. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃.

The term “heteroaryl,” as used herein, alone or in combination, refersto a 3 to 7 membered unsaturated heteromonocyclic ring, or a fusedmonocyclic, bicyclic, or tricyclic ring system in which at least one ofthe fused rings is aromatic, which contains at least one atom selectedfrom the group consisting of O, S, and N. In certain embodiments, saidheteroaryl will comprise from 5 to 7 carbon atoms. The term alsoembraces fused polycyclic groups wherein heterocyclic rings are fusedwith aryl rings, wherein heteroaryl rings are fused with otherheteroaryl rings, wherein heteroaryl rings are fused withheterocycloalkyl rings, or wherein heteroaryl rings are fused withcycloalkyl rings. Examples of heteroaryl groups include pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl,indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl,quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl,benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl,benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplarytricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyland the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated monocyclic, bicyclic, ortricyclic heterocyclic group containing at least one heteroatom as aring member, wherein each said heteroatom may be independently selectedfrom the group consisting of nitrogen, oxygen, and sulfur. In certainembodiments, said hetercycloalkyl will comprise from 1 to 4 heteroatomsas ring members. In further embodiments, said hetercycloalkyl willcomprise from 1 to 2 heteroatoms as ring members. In certainembodiments, said hetercycloalkyl will comprise from 3 to 8 ring membersin each ring. In further embodiments, said hetercycloalkyl will comprisefrom 3 to 7 ring members in each ring. In yet further embodiments, saidhetercycloalkyl will comprise from 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocycle” are intended to include sulfones,sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclicfused and benzo fused ring systems; additionally, both terms alsoinclude systems where a heterocycle ring is fused to an aryl group, asdefined herein, or an additional heterocycle group. Examples ofheterocycle groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl,dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl,dihydrobenzodioxinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl,benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3-dioxanyl,1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl,pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and thelike. The heterocycle groups may be optionally substituted unlessspecifically prohibited.

The term “hydrazinyl” as used herein, alone or in combination, refers totwo amino groups joined by a single bond, i.e., —N—N— and not embodiedin a ring.

The term “hydroxy,” as used herein, alone or in combination, refers to—OH.

The term “hydroxyalkyl,” as used herein, alone or in combination, refersto a hydroxy group attached to the parent molecular moiety through analkyl group.

The term “imino,” as used herein, alone or in combination, refers to═N—.

The term “iminohydroxy,” as used herein, alone or in combination, refersto ═N(OH) and ═N—O—.

The phrase “in the main chain” refers to the longest contiguous oradjacent chain of carbon atoms starting at the point of attachment of agroup to the compounds of any one of the formulas disclosed herein.

The term “isocyanato” refers to a —NCO group.

The term “isothiocyanato” refers to a —NCS group.

The phrase “linear chain of atoms” refers to the longest straight chainof atoms independently selected from carbon, nitrogen, oxygen andsulfur.

The term “lower,” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 to and including6 carbon atoms.

The term “lower aryl,” as used herein, alone or in combination, meansphenyl or naphthyl, which may be optionally substituted as provided.

The term “lower heteroaryl,” as used herein, alone or in combination,means either 1) monocyclic heteroaryl comprising five or six ringmembers, of which between one and four said members may be heteroatomsselected from the group consisting of O, S, and N, or 2) bicyclicheteroaryl, wherein each of the fused rings comprises five or six ringmembers, comprising between them one to four heteroatoms selected fromthe group consisting of O, S, and N.

The term “lower cycloalkyl,” as used herein, alone or in combination,means a monocyclic cycloalkyl having between three and six ring members.Lower cycloalkyls may be unsaturated. Examples of lower cycloalkylinclude cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “lower heterocycloalkyl,” as used herein, alone or incombination, means a monocyclic heterocycloalkyl having between threeand six ring members, of which between one and four may be heteroatomsselected from the group consisting of O, S, and N. Examples of lowerheterocycloalkyls include pyrrolidinyl, imidazolidinyl, pyrazolidinyl,piperidinyl, piperazinyl, and morpholinyl. Lower heterocycloalkyls maybe unsaturated.

The term “lower amino,” as used herein, alone or in combination, refersto —NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, lower alkyl, and lower heteroalkyl, any of whichmay be optionally substituted. Additionally, the R and R′ of a loweramino group may combine to form a five- or six-memberedheterocycloalkyl, either of which may be optionally substituted.

The term “mercaptyl” as used herein, alone or in combination, refers toan RS— group, where R is as defined herein.

The term “nitro,” as used herein, alone or in combination, refers to—NO₂.

The terms “oxy” or “oxa,” as used herein, alone or in combination, referto —O—.

The term “oxo,” as used herein, alone or in combination, refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein, alone or in combination, refersto an alkyl group where all of the hydrogen atoms are replaced byhalogen atoms.

The terms “sulfonate,” “sulfonic acid,” and “sulfonic,” as used herein,alone or in combination, refer the —SO₃H group and its anion as thesulfonic acid is used in salt formation.

The term “sulfanyl,” as used herein, alone or in combination, refers to—S—.

The term “sulfinyl,” as used herein, alone or in combination, refers to—S(O)—.

The term “sulfonyl,” as used herein, alone or in combination, refers to—S(O)₂—.

The term “N-sulfonamido” refers to a RS(═O)₂NR′— group with R and R′ asdefined herein.

The term “S-sulfonamido” refers to a —S(═O)₂NRR′, group, with R and R′as defined herein.

The terms “thia” and “thio,” as used herein, alone or in combination,refer to a —S— group or an ether wherein the oxygen is replaced withsulfur. The oxidized derivatives of the thio group, namely sulfinyl andsulfonyl, are included in the definition of thia and thio.

The term “thiol,” as used herein, alone or in combination, refers to an—SH group.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′— group, with R and R′as defined herein.

The term “0-thiocarbamyl” refers to a —OC(S)NRR′, group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

The term “trihalomethoxy” refers to a X₃CO— group where X is a halogen.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When a group is defined to be “null,” what is meant is that said groupis absent.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Twosubstituents may be joined together to form a fused five-, six-, orseven-membered carbocyclic or heterocyclic ring consisting of zero tothree heteroatoms, for example forming methylenedioxy or ethylenedioxy.An optionally substituted group may be unsubstituted (e.g., —CH₂CH₃),fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) orsubstituted at a level anywhere in-between fully substituted andmonosubstituted (e.g., —CH₂CF₃). Where substituents are recited withoutqualification as to substitution, both substituted and unsubstitutedforms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl and heterocycloalkyl, any of which may be optionallysubstituted. Such R and R′ groups should be understood to be optionallysubstituted as defined herein. Whether an R group has a numberdesignation or not, every R group, including R, R′ and R^(n) where n=(1,2, 3, . . . n), every substituent, and every term should be understoodto be independent of every other in terms of selection from a group.Should any variable, substituent, or term (e.g. aryl, heterocycle, R,etc.) occur more than one time in a formula or generic structure, itsdefinition at each occurrence is independent of the definition at everyother occurrence. Those of skill in the art will further recognize thatcertain groups may be attached to a parent molecule or may occupy aposition in a chain of elements from either end as written. Thus, by wayof example only, an unsymmetrical group such as —C(O)N(R)— may beattached to the parent moiety at either the carbon or the nitrogen.

Asymmetric centers exist in the compounds disclosed herein. Thesecenters are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the invention encompasses all stereochemical isomericforms, including diastereomeric, enantiomeric, and epimeric forms, aswell as d-isomers and 1-isomers, and mixtures thereof. Individualstereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein may exist as geometric isomers. The presentinvention includes all cis, trans, syn, anti, entgegen (E), and zusammen(Z) isomers as well as the appropriate mixtures thereof. Additionally,compounds may exist as tautomers; all tautomeric isomers are provided bythis invention. Additionally, the compounds disclosed herein can existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered to be part oflarger substructure. A bond may be single, double, or triple unlessotherwise specified. A dashed line between two atoms in a drawing of amolecule indicates that an additional bond may be present or absent atthat position.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

“PASK inhibitor” as used herein refers to a compound that exhibits an(IC₅₀/EC₅₀) with respect to PASK activity of no more than about 100 μMand more typically not more than about 50 μM, as measured in the PASKassay described generally hereinbelow. IC₅₀ is that concentration ofinhibitors which reduces the activity of PASK to half-maximal level.Certain compounds disclosed herein have been discovered to exhibitinhibition against PASK.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder.This amount will achieve the goal of reducing or eliminating the saiddisease or disorder.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. The term “patient” means all mammals includinghumans. Examples of patients include humans, cows, dogs, cats, goats,sheep, pigs, and rabbits. Preferably, the patient is a human.

The term “prodrug” refers to a compound that is made more active invivo. Certain compounds disclosed herein may also exist as prodrugs, asdescribed in Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M.Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs of the compoundsdescribed herein are structurally modified forms of the compound thatreadily undergo chemical changes under physiological conditions toprovide the compound. Additionally, prodrugs can be converted to thecompound by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to a compound when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent. Prodrugs are often useful because, in some situations, they maybe easier to administer than the compound, or parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present invention includes compounds listed above in the formof salts, including acid addition salts. Suitable salts include thoseformed with both organic and inorganic acids. Such acid addition saltswill normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate (p-tosylate), and undecanoate. Also, basic groupsin the compounds disclosed herein can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.Examples of acids which can be employed to form therapeuticallyacceptable addition salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, and citric. Salts can also be formed by coordinationof the compounds with an alkali metal or alkaline earth ion. Hence, thepresent invention contemplates sodium, potassium, magnesium, and calciumsalts of the compounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

While it may be possible for the compounds of the subject invention tobe administered as the raw chemical, it is also possible to present themas a pharmaceutical formulation. Accordingly, provided herein arepharmaceutical formulations which comprise one or more of certaincompounds disclosed herein, or one or more pharmaceutically acceptablesalts, esters, prodrugs, amides, or solvates thereof, together with oneor more pharmaceutically acceptable carriers thereof and optionally oneor more other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art; e.g., in Remington'sPharmaceutical Sciences. The pharmaceutical compositions disclosedherein may be manufactured in any manner known in the art, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject invention or a pharmaceutically acceptable salt, ester, amide,prodrug or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Formulations of the compounds disclosed herein suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein. All formulationsfor oral administration should be in dosages suitable for suchadministration. The push-fit capsules can contain the active ingredientsin admixture with filler such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. The formulations may be presentedin unit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in powder form or in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or sterile pyrogen-free water,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, polyethylene glycol, or otherglycerides.

Certain compounds disclosed herein may be administered topically, thatis by non-systemic administration. This includes the application of acompound disclosed herein externally to the epidermis or the buccalcavity and the instillation of such a compound into the ear, eye andnose, such that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral,intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as gels, liniments, lotions, creams,ointments or pastes, and drops suitable for administration to the eye,ear or nose. The active ingredient for topical administration maycomprise, for example, from 0.001% to 10% w/w (by weight) of theformulation. In certain embodiments, the active ingredient may compriseas much as 10% w/w. In other embodiments, it may comprise less than 5%w/w. In certain embodiments, the active ingredient may comprise from 2%w/w to 5% w/w. In other embodiments, it may comprise from 0.1% to 1% w/wof the formulation.

For administration by inhalation, compounds may be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form, in for example, capsules, cartridges,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator or insufflator.

Preferred unit dosage formulations are those containing an effectivedose, as herein below recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations described above may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

Compounds may be administered orally or via injection at a dose of from0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 2 g/day. Tablets or other forms of presentation provided indiscrete units may conveniently contain an amount of one or morecompounds which is effective at such dosage or as a multiple of thesame, for instance, units containing 5 mg to 500 mg, usually around 10mg to 200 mg.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The compounds can be administered in various modes, e.g. orally,topically, or by injection. The precise amount of compound administeredto a patient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. Also, the route of administrationmay vary depending on the condition and its severity.

In certain instances, it may be appropriate to administer at least oneof the compounds described herein (or a pharmaceutically acceptablesalt, ester, or prodrug thereof) in combination with another therapeuticagent. By way of example only, if one of the side effects experienced bya patient upon receiving one of the compounds herein is hypertension,then it may be appropriate to administer an anti-hypertensive agent incombination with the initial therapeutic agent. Or, by way of exampleonly, the therapeutic effectiveness of one of the compounds describedherein may be enhanced by administration of an adjuvant (i.e., by itselfthe adjuvant may only have minimal therapeutic benefit, but incombination with another therapeutic agent, the overall therapeuticbenefit to the patient is enhanced). Or, by way of example only, thebenefit of experienced by a patient may be increased by administeringone of the compounds described herein with another therapeutic agent(which also includes a therapeutic regimen) that also has therapeuticbenefit. By way of example only, in a treatment for diabetes involvingadministration of one of the compounds described herein, increasedtherapeutic benefit may result by also providing the patient withanother therapeutic agent for diabetes. In any case, regardless of thedisease, disorder or condition being treated, the overall benefitexperienced by the patient may simply be additive of the two therapeuticagents or the patient may experience a synergistic benefit.

Specific, non-limiting examples of possible combination therapiesinclude use of a compound as disclosed herein, and at least one otheragent selected from the group comprising:

-   -   a) anti-diabetic agents such as insulin, insulin derivatives and        mimetics; insulin secretagogues such as the sulfonylureas, e.g.,        Glipizide, glyburide and Amaryl; insulinotropic sulfonylurea        receptor ligands such as meglitinides, e.g., nateglinide and        repaglinide; insulin sensitizer such as protein tyrosine        phosphatase-1B (PTP-1B) inhibitors such as PTP-112; GSK3        (glycogen synthase kinase-3) inhibitors such as SB-517955,        SB-4195052, SB-216763, NN-57-05441 and NN-57-05445; RXR ligands        such as GW-0791 and AGN-194204; sodium-dependent glucose        co-transporter inhibitors such as T-1095; glycogen phosphorylase        A inhibitors such as BAY R3401; biguanides such as metformin;        alpha-glucosidase inhibitors such as acarbose; GLP-1 (glucagon        like peptide-1), GLP-1 analogs such as Exendin-4 and GLP-1        mimetics; DPPIV (dipeptidyl peptidase IV) inhibitors such as        DPP728, LAF237 (vildagliptin—Example 1 of WO 00/34241), MK-0431,        saxagliptin, GSK23A; an AGE breaker; a thiazolidinedione        derivative (glitazone) such as pioglitazone or rosiglitazone;        and a non-glitazone type PPARδ agonist e.g. GI-262570;    -   b) hypolipidemic agents such as 3-hydroxy-3-methyl-glutaryl        coenzyme A (HMG-CoA) reductase inhibitors, e.g., lovastatin,        pitavastatin, simvastatin, pravastatin, cerivastatin,        mevastatin, velostatin, fluvastatin, dalvastatin, atorvastatin,        rosuvastatin and rivastatin; squalene synthase inhibitors; FXR        (farnesoid×receptor) and LXR (liver×receptor) ligands;        cholestyramine; fibrates; nicotinic acid and aspirin;    -   c) an anti-obesity agent or appetite regulating agent such as        phentermine, leptin, bromocriptine, dexamphetamine, amphetamine,        fenfluramine, dexfenfluramine, sibutramine, orlistat,        dexfenfluramine, mazindol, phentermine, phendimetrazine,        diethylpropion, fluoxetine, bupropion, topiramate,        diethylpropion, benzphetamine, phenylpropanolamine or ecopipam,        ephedrine, pseudoephedrine or cannabinoid receptor antagonists;    -   d) anti-hypertensive agents, e.g., loop diuretics such as        ethacrynic acid, furosemide and torsemide; diuretics such as        thiazide derivatives, chlorothiazide, hydrochlorothiazide,        amiloride; angiotensin converting enzyme (ACE) inhibitors such        as benazepril, captopril, enalapril, fosinopril, lisinopril,        moexipril, perinodopril, quinapril, ramipril and trandolapril;        inhibitors of the Na-K-ATPase membrane pump such as digoxin;        neutral endopeptidase (NEP) inhibitors e.g. thiorphan,        terteo-thiorphan, SQ29072; ECE inhibitors e.g. SLV306; ACE/NEP        inhibitors such as omapatrilat, sampatrilat and fasidotril;        angiotensin n antagonists such as candesartan, eprosartan,        irbesartan, losartan, tehnisartan and valsartan, in particular        valsartan; renin inhibitors such as aliskiren, terlakiren,        ditekiren, RO 66-1132, RO-66-1168; □-adrenergic receptor        blockers such as acebutolol, atenolol, betaxolol, bisoprolol,        metoprolol, nadolol, propranolol, sotalol and timolol; inotropic        agents such as digoxin, dobutamine and milrinone; calcium        channel blockers such as amlodipine, bepridil, diltiazem,        felodipine, nicardipine, nimodipine, nifedipine, nisoldipine and        verapamil; aldosterone receptor antagonists; and aldosterone        synthase inhibitors;    -   e) an HDL increasing compound;    -   f) cholesterol absorption modulator such as etizimibe and        KT6-971;    -   g) Apo-Al analogues and mimetics;    -   h) thrombin inhibitors such as Ximelagatran;    -   i) aldosterone inhibitors such as anastrazole, fadrazole, and        eplerenone;    -   j) inhibitors of platelet aggregation such as aspirin, and        clopidogrel bisulfate;    -   k) estrogen, testosterone, a selective estrogen receptor        modulator, and a selective androgen receptor modulator;    -   l) a chemotherapeutic agent such as aromatase inhibitors e.g.        femara, anti-estrogens, topoisomerase I inhibitors,        topoisomerase II inhibitors, microtubule active agents,        alkylating agents, antineoplastic antimetabolites, platin        compounds, and compounds decreasing the protein kinase activity        such as a PDGF receptor tyrosine kinase inhibitor such as        miatinib; and    -   m) an agent interacting with a 5-HT3 receptor and/or an agent        interacting with 5-HT4 receptor such as tegaserod described in        the U.S. Pat. No. 5,510,353 as example 13, tegaserod hydrogen        maleate, cisapride, and cilansetron.

In any case, the multiple therapeutic agents (at least one of which is acompound disclosed herein) may be administered in any order or evensimultaneously. If simultaneously, the multiple therapeutic agents maybe provided in a single, unified form, or in multiple forms (by way ofexample only, either as a single pill or as two separate pills). One ofthe therapeutic agents may be given in multiple doses, or both may begiven as multiple doses. If not simultaneous, the timing between themultiple doses may be any duration of time ranging from a few minutes tofour weeks.

Thus, in another aspect, certain embodiments provide methods fortreating PASK-mediated disorders in a human or animal subject in need ofsuch treatment comprising administering to said subject an amount of acompound disclosed herein effective to reduce or prevent said disorderin the subject, optionally in combination with at least one additionalagent that is known in the art. In a related aspect, certain embodimentsprovide therapeutic compositions comprising at least one compounddisclosed herein in combination with one or more additional agents forthe treatment of PASK-mediated disorders.

Recent studies have found that elevated medium glucose concentrationscaused post-translational activation of PASK. It has also beendemonstrated that PASK activity is required for glucose-stimulatedinsulin expression, as shown by studies in PASK1 mice. It has also beendemonstrated that PASK deletion results in nearly complete resistance tothe phenotypes caused by a high-fat diet, including obesity, insulinresistance and hepatic fat accumulation. It has been postulated thatthis protection may be due to an increase in AMPK expression in each ofthe relevant tissues. PASK deletion abrogates nearly all of themaladaptive phenotype associated with a high-fat diet, possibly in partvia maintenance of AMPK expression. Increasing AMPK signaling is aproven therapeutic strategy, as illustrated by Metformin, which acts byincreasing the phosphorylation and activation of AMPK. Inhibition ofPASK signaling elicits similar beneficial effects, but through adistinct mechanism. This complementary therapeutic strategy, eitheralone or in combination, can be efficacious in the treatment ofmetabolic diseases. In any case, it appears that PASK inhibition canprovide an effective therapeutic strategy for the treatment of diseases,for example Type 2 diabetes, insulin resistance in general, and themetabolic syndrome.

Metabolic syndrome (also known as metabolic syndrome X) is characterizedby having at least three of the following symptoms: insulin resistance;abdominal fat—in men this is defined as a 40 inch waist or larger, inwomen 35 inches or larger; high blood sugar levels—at least 110milligrams per deciliter (mg/dL) after fasting; high triglycerides—atleast 150 mg/dL in the blood stream; low HDL—less than 40 mg/dL;pro-thrombotic state (e.g. high fibrinogen or plasminogen activatorinhibitor in the blood); or blood pressure of 130/85 mmHg or higher. Aconnection has been found between metabolic syndrome and otherconditions such as obesity, high blood pressure and high levels of LDLcholesterol, all of which are risk factors for cardiovascular diseases.For example, an increased link between metabolic syndrome andatherosclerosis has been shown. People with metabolic syndrome are alsomore prone to developing type 2 diabetes, as well as PCOS (polycysticovarian syndrome) in women and prostate cancer in men.

As described above, insulin resistance can be manifested in severalways, including type 2 diabetes. Type 2 diabetes is the condition mostobviously linked to insulin resistance. Compensatory hyperinsulinemiahelps maintain normal glucose levels—often for decades, before overtdiabetes develops. Eventually the beta cells of the pancreas are unableto overcome insulin resistance through hypersecretion. Glucose levelsrise, and a diagnosis of diabetes can be made. Patients with type 2diabetes remain hyperinsulinemic until they are in an advanced stage ofdisease. As described above, insulin resistance can also correlate withhypertension. One half of patients with essential hypertension areinsulin resistant and hyperinsulinemic, and there is evidence that bloodpressure is linked to the degree of insulin resistance. Hyperlipidemia,too, is associated with insulin resistance. The lipid profile ofpatients with type 2 diabetes includes increased serum very-low-densitylipoprotein cholesterol and triglyceride levels and, sometimes, adecreased low-density lipoprotein cholesterol level. Insulin resistancehas been found in persons with low levels of high-density lipoprotein.Insulin levels have also been linked to very-low-density lipoproteinsynthesis and plasma triglyceride levels.

Accordingly, also disclosed are methods of treating insulin resistancein a subject comprising selecting a subject in need of treatment forinsulin resistance; and administering to the subject an effective amountof a compound that inhibits PASK.

Specific diseases to be treated by the compounds, compositions, andmethods disclosed herein are those mediated at least in part by PASK.Accordingly, disclosed herein are methods: for reducing glycogenaccumulation in a subject; for raising HDL or HDLc, lowering LDL orLDLc, shifting LDL particle size from small dense to normal LDL,lowering VLDL, lowering triglycerides, or inhibiting cholesterolabsorption in a subject; for reducing insulin resistance, enhancingglucose utilization or lowering blood pressure in a subject; forreducing visceral fat in a subject; for reducing serum transaminases ina subject; or for treating disease; all comprising the administration ofa therapeutic amount of a compound as described herein, to a patient inneed thereof. In further embodiments, the disease to be treated may be ametabolic disease. In further embodiment, the metabolic disease may beselected from the group consisting of: obesity, diabetes melitus,especially Type 2 diabetes, hyperinsulinemia, glucose intolerance,metabolic syndrome X, dyslipidemia, hypertriglyceridemia,hypercholesterolemia, and hepatic steatosis. In other embodiments, thedisease to be treated may be selected from the group consisting of:cardiovascular diseases including vascular disease, atherosclerosis,coronary heart disease, cerebrovascular disease, heart failure andperipheral vessel disease. In preferred embodiments, the methods abovedo not result in the induction or maintenance of a hypoglycemic state.

Additionally, the PASK modulators disclosed herein may be used to treatproliferative disorders such as cancers. Hematological andnon-hematological cancers which may be treated or prevented include butare not limited to multiple myeloma, acute and chronic leukemiasincluding Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia(CLL), and Chronic Myelogenous Leukemia (CLL), lymphomas, includingHodgkin's lymphoma and non-Hodgkin's lymphoma (low, intermediate, andhigh grade), malignancies of the brain, head and neck, breast, lung,reproductive tract, upper digestive tract, pancreas, liver, renal,bladder, prostate and colon/rectum.

Besides being useful for human treatment, certain compounds andformulations disclosed herein may also be useful for veterinarytreatment of companion animals, exotic animals and farm animals,including mammals, rodents, and the like. More preferred animals includehorses, dogs, and cats.

REFERENCES CITED

The following is a list of references cited herein which, while notnecessarily comprehensive, is provided for the convenience of thereader. All references, patents, and patent applications cited hereinare hereby incorporated by reference as if written herein in theirentireties. When the teachings of these references contradict theteachings presented expressly herein, the present disclosure controls.

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The invention is further illustrated by the following examples, whichcan be made by the methods described herein or by one skilled in the artwithout undue experimentation, or can be purchased from commercialsources.

EXAMPLE 15-(Benzo[d][1,3]dioxol-5-yl)-2-benzyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-benzyl-1H-1,2,4-triazol-5-amine (100 mg, 0.57 mmol)in n-BuOH (3 ml) was added ethyl3-(2H-1,3-benzodioxol-5-yl)-3-oxopropanoate (200 mg, 0.85 mmol) and4-methylbenzene-1-sulfonic acid (4 mg, 0.02 mmol). After stirringovernight at reflux, the solids were collected by filtration and washedwith ethyl acetate (3×10 mL), methanol (3×10 ml) and dried to give5-(benzo[d][1,3]dioxol-5-yl)-2-benzyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a white solid (50.2 mg, 25%).

LC/MS (ES, m/z): [M+H]⁺ 347.05

¹H NMR (300 MHz, DMSO) δ 7.43-7.56 (m, 2H), 7.18-7.35 (m, 5H), 7.05 (d,J=8.1 Hz, 1H), 6.28 (s, 1H), 6.12 (s, 2H), 4.10 (s, 2H)

EXAMPLE 25-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (100 mg, 0.57mmol) in diphenyl ether (3 ml) was added ethyl3-(1H-1,2,3-benzotriazol-5-yl)-3-oxopropanoate (300 mg, 1.29 mmol) and4-methylbenzene-1-sulfonic acid (5 mg, 0.02 mmol). After stirring 1 h at170° C., the solids were collected by filtration, washed with ethylacetate (2×10 ml), methanol (3×10 ml) and dried to give5-(1H-benzo[d][1,2,3]triazol-5-yl)-2-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a off-white solid (36.7 mg, 18%).

LC/MS (ES, m/z): [M+H]⁺ 331.0

¹H NMR (300 MHz, DMSO) δ 8.70 (s, 1H), 8.40 (s, 1H), 8.22 (d, J=7.8 Hz,1H), 7.73-7.97 (m, 3H), 7.443-7.47 (t, J=6.9 Hz, 1H), 6.25 (s, 1H)

EXAMPLE 35-(1H-Indazol-5-yl)-2-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 1H-Indazole-5-carboxylic acid

To a solution of methyl 1H-indazole-5-carboxylate (10 g, 56.82 mmol) inmethanol (80 ml) was added a solution of sodium hydroxide (9.09 g,227.28 mmol) in water (30 ml) with stirring overnight at 60° C. in anoil bath. The resulting mixture was concentrated under vacuum, thenquenched by the addition of water (30 ml), and the pH adjusted to 6 withHCl (3N). The solids were collected by filtration to afford1H-indazole-5-carboxylic acid as a yellow solid (8.6 g, 93%). LC/MS (ES,m/z): [M+H]⁺ 163.0

Step 2. 1-Acetyl-1H-indazole-5-carboxylic acid

To a solution of 1H-indazole-5-carboxylic acid (8.6 g, 53.04 mmol) inAcOH (200 ml) was added acetic anhydride (16.2 g), and the reaction wasstirred for 2.5 h at 80° C. The resulting solution was diluted withn-hexane (400 ml). The solids were collected by filtration and washedwith hexane (5×100 ml) to afford 1-acetyl-1H-indazole-5-carboxylic acidas an orange solid (8.5 g, 78%).

LC/MS (ES, m/z): [M+H]⁺ 205.0

¹H NMR (300 MHz, DMSO) δ 13.09 (s, 1H), 8.60 (d, J=0.6 Hz, 1H),8.52-8.53 (m, 1H), 8.37 (d, J=8.7 Hz, 1H), 8.17-8.20 (m, 1H), 2.75 (s,3H)

Step 3. Ethyl 3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate

To a solution of 1-acetyl-1H-indazole-5-carboxylic acid (8.5 g, 41.63mmol) in tetrahydrofuran (200 ml) was added CDI (20 g), and theresulting mixture was stirred for 2.5 h at 40° C., and then addeddropwise to a solution of the magnesium salt of malonic acid monoethylester [prepared via the addition of Et₃N (14 g, 138.25 mmol) and MgCl₂(19 g, 200 mmol) to a solution of potassium monoethylonate (24 g, 147.17mmol) in acetonitrile (200 ml)] followed by stirring at room temperaturefor 2.5 h at 0° C. The resulting solution was stirred for an additional2.5 h at 80° C. The reaction was then quenched by the addition of water(500 ml) and the pH adjusted to 3 with HCl (3N). The solids werecollected by filtration and washed with water (3×50 ml) to afford ethyl3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate as a light yellow solid(8.6 g, 75%).

¹H NMR (300 MHz, CDCl₃) δ 8.47-8.56 (m, 1H), 8.41 (d, J=0.9 Hz, 1H),8.20 (d, J=1.5 Hz, 1H), 8.16-8.19 (m, 1H), 4.21-4.34 (m, 2H), 4.09 (s,2H), 2.83 (s, 3H), 1.25-1.30 (t, J=7.2 Hz, 3H)

Step 4.5-(1-Acetyl-1H-indazol-5-yl)-2-(pyridin-2-yl)-4H,7H-[1,2,4]triazolo[1,5-a]pyrimidin-7-one

To a solution of 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (150 mg, 0.93mmol) in diphenyl ether (3 mL) was added ethyl3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate (510 mg, 1.86 mmol) andTsOH (8 mg, 0.05 mmol), and the mixture was stirred for 1 h at 170° C.The resulting solution was diluted with n-hexane (8 ml), and the productwas collected by filtration and washed with methanol (2 ml) to afford5-(1-acetyl-1H-indazol-5-yl)-2-(pyridin-2-yl)-4H,7H-[1,2,4]triazolo[1,5-a]pyrimidin-7-oneas a brown crude solid (120 mg, crude).

Step 5.5-(1H-Indazol-5-yl)-2-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of5-(1-acetyl-1H-indazol-5-yl)-2-(pyridin-2-yl)-4H,7H-[1,2,4]triazolo[1,5-a]pyrimidin-7-one(120 mg, crude) in methanol (10 ml) was added a solution of potassiumcarbonate (48 mg, 0.35 mmol) in water (1 ml), and the reaction wasstirred overnight at room temperature. The resulting solution wasconcentrated and diluted with water (5 ml). The solids were collected byfiltration to give a crude product, which was purified byFlash-Prep-HPLC to afford5-(1H-indazol-5-yl)-2-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a brown solid (36.9 mg, 12% 2 steps).

LC/MS (ES, m/z): [M+H]⁺ 330.0

¹H NMR (300 MHz, DMSO) δ 13.33 (s, 1H), 8.76 (d, J=3.9 Hz, 1H), 8.41 (s,1H), 8.09-8.25 (m, 2H), 7.99-8.04 (m, 2H), 7.86-7.93 (m, 2H), 7.48-7.71(m, 2H), 6.41 (s, 1H)

EXAMPLE 45-(1H-indazol-5-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 3-Methyl-1H-1,2,4-triazol-5-amine

To a solution of (diaminomethyl)hydrazine carbonic acid salt (11 g, 80.3mmol) in water (30 ml) was added dropwise AcOH (9.6 g, 160 mmol) at roomtemperature. The pH was adjusted 4 with HNO₃ (0.1 ml, conc), and thereation mixture was stirred for 45 h at reflux. The resulting mixturewas concentrated under vacuum to give a residue, which was purified by asilica gel column (2%-20% methanol in dichloromethane) to afford3-methyl-1H-1,2,4-triazol-5-amine as a white solid (4 g, 50%).

LC/MS (ES, m/z): [M+H]⁺ 99.0

¹H NMR (300 MHz, DMSO) δ 1.84 (s, 3H)

Step 2.5-(1-Acetyl-1H-indazol-5-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-methyl-1H-1,2,4-triazol-5-amine (150 mg, 1.52 mmol)in diphenyl ether (2 ml) was added ethyl3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate (710 mg, 2.59 mmol), TsOH(11 mg, 0.06 mmol), and the mixture was stirred for 1 h at 170° C. Theresulting solution was diluted with petroleum ether (8 ml), and solidswere collected by filtration and washed with ethyl ether (3 ml) toafford5-(1-acetyl-1H-indazol-5-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a crude brown solid (100 mg, crude).

LC/MS (ES, m/z): [M+H]⁺ 309.0

Step 3.5-(1H-Indazol-5-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of5-(1-acetyl-1H-indazol-5-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one(100 mg, crude) in methanol (3 ml) was added a solution of potassiumcarbonate (45 mg, 0.33 mmol) in water (1 ml), and the mixture wasstirred overnight at room temperature. The resulting solution wasconcentrated under vacuum to give a residue, which was diluted withwater (5 ml). The solids were collected by filtration and purified byPrep-HPLC to afford5-(1H-indazol-5-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a white solid (28.7 mg, 7% 2 steps).

LC/MS (ES, m/z): [M+H]⁺ 267.0

¹H NMR (300 MHz, DMSO) δ 13.27 (s, 1H), 8.33 (s, 1H), 8.22 (s, 1H),7.84-7.89 (m, 1H), 7.64 (d, J=8.4 Hz, 1H), 6.38 (s, 1H), 2.42 (s, 3H)

EXAMPLE 52-Ethyl-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1.5-(1-Acetyl-1H-indazol-5-yl)-2-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-ethyl-1H-1,2,4-triazol-5-amine (150 mg, 1.34 mmol) indiphenyl ether (2 ml) was added ethyl3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate (550 mg, 2.00 mmol) andTsOH (8 mg, 0.04 mmol), and the reaction mixture was stirred for 1 h at170° C. The solids were collected by filtration and washed with hexane(10 ml) and ethanol (3 ml) to afford5-(1-acetyl-1H-indazol-5-yl)-2-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a yellow solid (130 mg, crude).

LC/MS (ES, m/z): [M+H]⁺ 323.0

Step 2.2-Ethyl-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of5-(1-acetyl-1H-indazol-5-yl)-2-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one(130 mg, crude) in methanol (5 ml) was added potassium carbonate (20 mg,0.14 mmol) and water (1 ml), and the reaction mixture was stirredovernight at room temperature. The reaction mixture was concentratedunder vacuum and diluted with water (10 ml). The product was collectedby filtration and washed with ethanol (10 ml) to afford2-ethyl-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one asa yellow solid (66 mg, 18% 2 steps).

LC/MS (ES, m/z): [M+H]⁺ 281.0

¹H NMR (300 MHz, DMSO) δ 8.39 (s, 1H), 8.23-8.32 (m, 1H), 7.88-7.96 (m,1H), 7.64 (d, J=8.7 Hz, 1H), 6.39 (s, 1H), 2.74-2.86 (m, 2H), 1.28-1.33(t, J=4.5 Hz, 3H)

EXAMPLE 65-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-ethyl-1H-1,2,4-triazol-5-amine (150 mg, 0.89 mmol) indiphenyl ether (2 ml) was added ethyl3-(1H-benzo[d][1,2,3]triazol-5-yl)-3-oxopropanoate (500 mg, 2.15 mmol)and TsOH (8 mg, 0.04 mmol), and the reaction mixture was stirredovernight at 130° C. The solids were collected by filtration and washedwith methanol (20 ml) to afford5-(1H-benzo[d][1,2,3]triazol-5-yl)-2-ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a off-white solid (66.4 mg, 18%).

LC/MS (ES, m/z): [M+H]⁺ 282.0

¹H NMR (300 MHz, DMSO) δ 8.50-8.57 (m, 1H), 7.90-8.15 (m, 2H), 6.40 (s,1H), 2.66-2.77 (m, 2H), 1.27-1.32 (t, J=7.5 Hz, 3H)

EXAMPLE 75-(1H-Benzo[d][1,2,3]triazol-5-yl)-N-methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide

Step 1. 2-(2-Carbamimidoylhydrazinyl)-2-oxoacetic acid

To a solution of 1-aminoguanidine carbonic acid salt (12 g, 88 mmol) inpyridine (50 ml) was added ethyl 2-chloro-2-oxoacetate (10 g, 73.24mmol). The resulting solution was stirred for 12 h at room temperature.The resulting solution was diluted with PE (200 ml). The solids werecollected by filtration and then dissolved in water (50 ml), the pH wasadjusted to 8 with NaOH (aq., 10N). The product precipitated and wasfiltered to afford ethyl 2-(2-carbamimidoylhydrazinyl)-2-oxoacetic acidas a white solid (2 g, 19%).

LC/MS (ES, m/z): [M+H]⁺ 147.0

Step 2. 5-Amino-1H-1,2,4-triazole-3-carboxylic acid

A solution of ethyl 2-(2-carbamimidoylhydrazinyl)-2-oxoacetic acid (2 g,35.60 mmol) in water (100 ml) was stirred for 12 h at 100° C. Theresulting mixture was concentrated under vacuum to give a residue, whichwas precipitated from water (10 ml) to afford5-amino-1H-1,2,4-triazole-3-carboxylic acid as a white solid (1.1 g,63%).

LC/MS (ES, m/z): [M+H]⁺ 129.0

Step 3. Methyl 5-amino-1H-1,2,4-triazole-3-carboxylate

To a solution of 5-amino-1H-1,2,4-triazole-3-carboxylic acid (3.9 g,30.45 mmol) in methanol (100 ml) was added SOCl₂ (12 g, 100.84 mmol).The resulting solution was stirred for 48 h at 70° C. The resultingmixture was concentrated under vacuum, dissolved in water (100 ml), thepH was adjusted to 8 with NaHCO₃ solution, and then extracted withdichloromethane (3×50 ml). The organic layer was dried over anhydrousmagnesium sulfate and concentrated under vacuum to afford methyl5-amino-1H-1,2,4-triazole-3-carboxylate as a white solid (800 mg, 65%).

LC/MS (ES, m/z): [M+H]⁺ 143.0

¹H NMR (300 MHz, DMSO): δ, 3.85 (s, 3H)

Step 4. 5-Amino-N-methyl-1H-1,2,4-triazole-3-carboxamide

A mixture of methyl 5-amino-1H-1,2,4-triazole-3-carboxylate (800 mg,5.63 mmol) and methylamine ethanol solution (30%, 10 ml) was stirred for3 h at 80° C. The resulting mixture was concentrated under vacuum toafford 5-amino-N-methyl-1H-1,2,4-triazole-3-carboxamide as a white solid(0.5 g, 63%).

LC/MS (ES, m/z): [M+H]⁺ 142.0

Step 5.5-(1H-Benzo[d][1,2,3]triazol-5-yl)-N-methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide

To a solution of 5-amino-N-methyl-1H-1,2,4-triazole-3-carboxamide (100mg, 0.71 mmol) in n-BuOH (3 ml) was added TsOH (4.8 mg, 0.03 mmol),ethyl 3-(1H-benzo[d][1,2,3]triazol-5-yl)-3-oxopropanoate (230 mg, 0.88mmol). The resulting solution was stirred for 12 h at reflux. The solidswere collected by filtration to afford5-(1H-benzo[d][1,2,3]triazol-5-yl)-N-methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamideas a white solid (39.8 mg, 18%).

LC/MS (ES, m/z): [M+H]⁺ 311.0

¹H NMR (300 MHz, DMSO) δ 8.46 (s, 1H), 8.02-8.10 (m, 1H), 7.85 (d, J=8.7Hz, 1H), 6.29 (s, 1H), 2.80 (s, 3H)

EXAMPLE 85-(4-Chloro-3-methoxyphenyl)-2-(4-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(4-chlorophenyl)-1H-1,2,4-triazol-5-amine (100 mg,0.51 mmol) in n-BuOH (1 ml) was added ethyl3-(4-chloro-3-methoxyphenyl)-3-oxopropanoate (200 mg, 0.78 mmol) andTsOH (5 mg, 0.02 mmol), and the resulting mixture was stirred overnightat 130° C. The solids were collected by filtration and washed withmethanol (2×5 ml) to afford5-(4-chloro-3-methoxyphenyl)-2-(4-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a light yellow solid (56.6 mg, 28%).

LC/MS (ES, m/z): [M+H]⁺ 387.0

¹H NMR (300 MHz, DMSO) δ 8.13 (d, J=8.7 Hz, 1H), 7.61-7.65 (m, 4H),7.49-7.52 (t, J=6.6 Hz, 1H), 6.50 (s, 1H), 4.00 (s, 3H)

EXAMPLE 95-(4-Chloro-3-methoxyphenyl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(4-fluorophenyl)-1H-1,2,4-triazol-5-amine (100 mg,0.56 mmol) in n-BuOH (3 ml) was added TsOH (5 mg, 0.03 mmol), and ethyl3-(4-chloro-3-methoxyphenyl)-3-oxopropanoate (200 mg, 0.78 mmol), andthe resulting mixture was stirred for 36 hours at 130° C. in an oilbath. The solids were collected by filtration to give a crude product,which was purified by Flash-Prep-HPLC to afford5-(4-chloro-3-methoxyphenyl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a white solid (33.5 mg, 16%).

LC/MS (ES, m/z): [M+H]⁺ 372.1

¹H NMR (300 MHz, DMSO) δ 8.15-8.20 (m, 2H), 7.80 (d, J=1.5 Hz, 1H),7.57-7.61 (m, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.30-7.36 (t, J=9.0 Hz, 2H),6.26 (s, 1H), 3.98 (s, 3H)

EXAMPLE 102-(3,4-Dichlorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 3,4-Dichlorobenzoyl chloride

A solution of 3,4-dichlorobenzoic acid (20 g, 105.26 mmol) in thionylchloride (200 ml) was stirred overnight at 90° C. The resulting mixturewas concentrated under vacuum to afford 3,4-dichlorobenzoyl chloride aslight yellow oil (20 g, 91%).

Step 2. 2-(3,4-Dichlorobenzoyl)hydrazinecarboximidamide

To a solution of hydrazinecarboximidamide carbonic acid salt (15.6 g,114.61 mmol) in pyridine (100 ml) was added 3,4-dichlorobenzoyl chloride(20 g, 95.48 mmol), and the resulting mixture was stirred overnight atroom temperature, then concentrated under vacuum to give a residue,which was dissolved in water (30 ml) and adjusted to pH 12 with sodiumhydroxide (aq., sat). The product precipitated and was collected byfiltration and dried in an oven under reduced pressure to afford2-(3,4-dichlorobenzoyl)hydrazinecarboximidamide as a light yellow solid(9.5 g, 40%).

LC/MS LC/MS (ES, m/z): [M+H]⁺ 247.0

Step 3. 3-(3,4-Dichlorophenyl)-1H-1,2,4-triazol-5-amine

A solution of 2-(3,4-dichlorobenzoyl)hydrazinecarboximidamide (9.5 g,38.62 mmol) in water (150 ml) was stirred for 7 h at reflux. The solidswere collected by filtration to afford3-(3,4-dichlorophenyl)-1H-1,2,4-triazol-5-amine as an off-white solid(7.9 g, 89%).

LC/MS (ES, m/z): [M+H]⁺ 229.0

¹H NMR (300 MHz, DMSO): δ 12.26 (s, 1H), 8.00-8.09 (m, 1H), 7.81-7.89(m, 1H), 7.65-7.77 (m, 1H), 6.17 (s, 2H)

Step 4.2-(3,4-Dichlorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(3,4-dichlorophenyl)-1H-1,2,4-triazol-5-amine (100mg, 0.44 mmol) in n-BuOH (1 ml) was added ethyl3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate (300 mg, 1.00 mmol) andTsOH (5 mg, 0.03 mmol), and the resulting mixture was stirred for 36 hat 130° C. The solids were collected by filtration and washed withmethanol (10 ml) to afford2-(3,4-dichlorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a light yellow solid (29 mg, 17%).

LC/MS LC/MS (ES, m/z): [M+H]⁺ 395.05

¹H NMR (300 MHz, DMSO): δ 13.31 (s, 1H), 8.39 (s, 1H), 8.23-8.27 (m,2H), 8.08-8.12 (m, 1H), 7.90 (d, J=7.5 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H),7.65 (d, J=8.7 Hz, 1H), 6.32 (s, 1H)

EXAMPLE 115-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-(3,4-dichlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(3,4-dichlorophenyl)-1H-1,2,4-triazol-5-amine (100mg, 0.44 mmol) in n-BuOH (1 ml) was added ethyl3-(1H-benzo[d][1,2,3]triazol-5-yl)-3-oxopropanoate (300 mg, 1.28 mmol)and TsOH (5 mg, 0.03 mmol), and the resulting mixture was stirred for 24h at 130° C. The solids were collected by filtration and washed withmethanol (10 ml) to afford5-(1H-benzo[d][1,2,3]triazol-5-yl)-2-(3,4-dichlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a light yellow solid (70.2 mg, 40%).

LC/MS LC/MS (ES, m/z): [M+H]⁺ 396.05

¹H NMR (300 MHz, DMSO): δ 8.37 (s, 1H), 8.30 (d, J=1.8 Hz, 1H),8.09-8.13 (m, 1H), 7.75-7.85 (m, 3H), 6.22 (s, 1H)

EXAMPLE 122-(3-Chlorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 3-Chlorobenzoyl chloride

A solution of 3-chlorobenzoic acid (30 g, 191.61 mmol) in thionylchloride (200 ml) was stirred overnight at 90° C. The resulting mixturewas concentrated under vacuum to afford 3-chlorobenzoyl chloride aslight yellow oil (30 g, 90%).

Step 2. 2-(3-Chlorobenzoyl)hydrazinecarboximidamide

To a solution of hydrazinecarboximidamide carbonic acid salt (14 g,241.12 mmol) in pyridine (100 ml) was added 3-chlorobenzoyl chloride (15g, 85.71 mmol), and the reaction mixture was stirred for 10 h at roomtemperature. The resulting mixture was concentrated under vacuum to givea residue, which was dissolved in water (30 ml) and adjusted to pH 12with sodium hydroxide (aq., sat.). The product was precipitated andcollected by filtration and dried in an oven under reduced pressure toafford 2-(3-chlorobenzoyl)hydrazinecarboximidamide as a light yellowsolid (7.8 g, 43%).

LC/MS LC/MS (ES, m/z): [M+H]⁺ 213.0

Step 3. 3-(3-Chlorophenyl)-1H-1,2,4-triazol-5-amine

A solution of 2-(3-chlorobenzoyl)hydrazinecarboximidamide (7.8 g, 36.68mmol) in water (150 ml) was stirred overnight at 90° C. The solids werecollected by filtration to afford3-(3-chlorophenyl)-1H-1,2,4-triazol-5-amine as a off-white solid (4.5 g,63%).

LC/MS (ES, m/z): [M+H]⁺ 195.05

¹H NMR (300 MHz, DMSO): δ 12.18 (s, 1H), 7.81-7.91 (m, 2H), 7.38-7.50(m, 2H), 6.13 (s, 1H)

Step 4.2-(3-Chlorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(3-chlorophenyl)-1H-1,2,4-triazol-5-amine (100 mg,0.52 mmol) in n-BuOH (1 ml) was added ethyl3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate (280 mg, 1.00 mmol) andTsOH (5 mg, 0.03 mmol), and the reaction mixture was stirred overnightat 130° C. The solids were collected by filtration and washed withmethanol (10 ml) to afford2-(3-chlorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a light yellow solid (75 mg, 34%).

LC/MS (ES, m/z): [M+H]⁺ 363.05

¹H NMR (300 MHz, DMSO): δ 8.65 (s, 1H), 8.09-8.28 (m, 4H), 7.48-7.61 (m,3H), 6.23 (s, 1H)

EXAMPLE 135-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-(3-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(3-chlorophenyl)-1H-1,2,4-triazol-5-amine (120 mg,0.62 mmol) in n-BuOH (1 mL) was added ethyl3-(1H-benzo[d][1,2,3]triazol-5-yl)-3-oxopropanoate (214 mg, 0.92 mmol)and TsOH (5.3 mg, 0.03 mmol), and the resulting mixture was stirred for5 h at 130° C. The solids were collected by filtration, washed with MeOH(3×1 mL) to afford5-(1H-benzo[d][1,2,3]triazol-5-yl)-2-(3-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a off-white solid (73 mg, 33%).

LC/MS (ES, m/z): [M+H]⁺ 364.0

¹H NMR (300 MHz, DMSO) δ 8.41 (s, 1H), 8.13 (m, 2H), 7.89 (d, J=8.80 Hz,1H), 7.78 (d, J=8.40 Hz, 1H), 7.54 (m, 2H), 6.24 (s, 1H)

EXAMPLE 145-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-isopropyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 3-Isopropyl-1H-1,2,4-triazol-5-amine

To a solution of isobutyric acid (25 g, 283.75 mmol) in water (25 mL)was added hydrazinecarboximidamide carbonic acid salt (15 g, 110.29mmol) and HNO₃ (0.3 mL), and the reaction mixture was stirred for 75 hat 110° C. The resulting mixture was concentrated under vacuum. Theresidue was applied onto a silica gel column (dichloromethane/methanol(15:1)) to afford 3-isopropyl-1H-1,2,4-triazol-5-amine as a white solid(9 g, 65%).

LC/MS (ES, m/z): [M+H]⁺ 127.0

¹H NMR (300 MHz, DMSO) δ2.88 (s, 1H), 1.27 (d, J=6.90 Hz, 6H)

Step 2.5-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-isopropyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-isopropyl-1H-1,2,4-triazol-5-amine (100 mg, 0.79mmol) in n-BuOH (1 mL) was added ethyl3-(1H-benzo[d][1,2,3]triazol-5-yl)-3-oxopropanoate (275 mg, 1.18 mmol)and TsOH (7 mg, 0.04 mmol), and the resulting mixture was stirred for 5h at 130° C. The solids were collected by filtration and washed withMeOH (3×1 mL) to afford5-(1H-benzo[d][1,2,3]triazol-5-yl)-2-isopropyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a off-white solid (45 mg, 19%).

LC/MS (ES, m/z): [M+H]⁺ 296.0

¹H NMR (300 MHz, DMSO) δ 8.34 (s, 1H), 7.84 (m, 1H), 7.73 (d, J=8.70 Hz,1H), 6.10 (s, 1H), 2.95 (m, 1H), 1.29 (d, J=6.90 Hz, 6H)

EXAMPLE 155-(Benzo[d][1,3]dioxol-5-yl)-2-(4-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 4-Chlorobenzoyl chloride

The solution of 4-chlorobenzoic acid (5 g, 31.94 mmol) in thionylchloride (150 ml) was stirred for 5 h at 80° C. and then concentratedunder vacuum to give 4-chlorobenzoyl chloride as a brown oil (4.5 g,crude).

Step 2. 2-(4-Chlorobenzoyl)hydrazinecarboximidamide

To a solution of hydrazinecarboximidamide bicarbonate salt (2.3 g, 31.05mmol) in pyridine (70 ml) was added dropwise 4-chlorobenzoyl chloride(4.5 g, crude) in DCM (20 ml). After stirring overnight at 15° C., theresulting mixture was concentrated under vacuum, quenched by theaddition of water (20 ml) and adjusted to pH 13 with sodium hydroxidesolution (10N). The product was collected by filtration and dried togive 2-(4-chlorobenzoyl)hydrazinecarboximidamide as a white solid (2.1g, crude). (ES, m/z): [M+H]⁺ 213.0

Step 3. 3-(4-Chlorophenyl)-1H-1,2,4-triazol-5-amine

A solution of 2-(4-chlorobenzoyl)hydrazinecarboximidamide (2.1 g) indiphenylether (15 ml) was stirred for 10 min at 170° C. The reaction wasthen quenched by the addition of hexane (30 ml) and the product wascollected by filtration, washed with hexane (3×10 ml) and dried in anoven under reduced pressure to give3-(4-chlorophenyl)-1H-1,2,4-triazol-5-amine as a light yellow solid (1g, 52%). (ES, m/z): [M+H]⁺ 194.0

¹H NMR (300 MHz, DMSO): δ 12.14 (s, 1H), 7.86-7.93 (t, J=8.7 Hz, 2H),7.44 (d, J=8.4 Hz, 2H), 6.10 (s, 2H)

Step 4.5-(Benzo[d][1,3]dioxol-5-yl)-2-(4-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(4-chlorophenyl)-1H-1,2,4-triazol-5-amine (50 mg,0.26 mmol) in diphenyether (2 ml) was added TsOH (2.2 mg, 0.01 mmol) andethyl 3-(benzo[d][1,3]dioxol-5-yl)-3-oxopropanoate (81 mg, 0.34 mmol).After stirring 2 h at 170° C., the reaction was quenched by the additionof hexane (5 ml), the product was collected by filtration and washedwith methanol (10 ml) and dried in an oven under reduced pressure togive5-(benzo[d][1,3]dioxol-5-yl)-2-(4-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a off-white solid (23.1 mg, 24%). (ES, m/z): [M+H]⁺ 367.0

¹H NMR (300 MHz, DMSO): δ 8.12 (d, J=8.4 Hz, 2H), 7.52-7.58 (m, 4H),6.95 (d, J=8.7 Hz, 1H), 6.07 (s, 1H), 6.06 (s, 2H)

EXAMPLE 165-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

A mixture of 5-methyl-2H-1,2,4-triazol-3-amine (147 mg, 1.50 mmol, 1.00equiv), acetic acid (3 mL), and ethyl3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-oxopropanoate (375 mg, 1.50mmol, 1.00 equiv) was stirred for 16 hr at 120° C., then concentrated todryness. The resulting solid was washed by 5 mL MeOH and collected byfiltration. The solid was dissolved in 10 mL of NH₄OH (aq.) andfiltered. The filtrate was adjusted to pH<5 with HOAc, then concentratedto dryness. The resulting solid was washed by 10 mL water, collected byfiltration, and dried to get 50 mg (12%) of5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a yellow solid.

LC-MS: (ES, m/z): 285 [M+H]⁺

¹H NMR (300 MHz, DMSO, ppm): 13.40 (s, 1H), 7.48-7.42 (m, 2H), 7.00 (s,1H), 6.31 (s, 1H), 4.31 (t, J=4.8 Hz, 4H), 2.41 (s, 3H)

EXAMPLE 175-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

A mixture of 2H-1,2,4-triazol-3-amine (200 mg, 2.38 mmol, 1.00 equiv),acetic acid (3 mL), and ethyl3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-oxopropanoate (600 mg, 2.40mmol, 1.01 equiv) was stirred for 24 hr at 120° C. and then concentratedto dryness. The resulting solid was washed with MeOH and collected byfiltration. The solid was dissolved in 15 mL of NH₄OH (aq.) andfiltered. The filtrate was adjusted to pH 5 with HOAc. The resultingsolid was collected by filtration and washed with water. The solid wasdried to get 95 mg (15%) of5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a white solid.

LC-MS: (ES, m/z): 271 [M+H]⁺

¹H NMR (300 MHz, DMSO, ppm) 13.45 (s, 1H), 7.47-7.40 (m, 2H), 7.02 (s,1H), 6.30 (s, 1H), 4.31 (t, J=4.8 Hz, 4H)

EXAMPLE 182-Benzyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

A mixture of 5-benzyl-2H-1,2,4-triazol-3-amine (174 mg, 1.00 mmol, 1.00equiv), acetic acid (3 mL), and ethyl3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-oxopropanoate (275 mg, 1.10mmol, 1.10 equiv) was stirred overnight at 110° C. The reaction wasconcentrated to dryness and purified with prep-TLC (MeOH:DCM=1:30) toafford 30.7 mg (8%) of2-benzyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a yellow solid.

LC-MS: (ES, m/z): 361 [M+H]⁺

¹H NMR (300 MHz, DMSO, ppm) 7.44-7.21 (m, 7H), 6.98 (d, J=8.4 Hz, 1H),6.20 (s, 1H), 4.29 (s, 4H), 4.07 (s, 2H)

EXAMPLE 192-Benzyl-5-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

A mixture of 5-benzyl-2H-1,2,4-triazol-3-amine (174 mg, 1.00 mmol, 1.00equiv), acetic acid (3 mL), and ethyl3-(4-methoxyphenyl)-3-oxopropanoate (228 mg, 1.10 mmol, 1.10 equiv) wasstirred overnight at 110° C. and then concentrated to dryness. Theresulting solid was washed with 5 mL MeOH and collected by filtration.The solid was dissolved in 10 mL of NH₄OH (aq.) and filtered. Thefiltrate was adjusted to pH 6 with HOAc. The resulting solid wascollected by filtration and washed with water. The solid was dried toget 26 mg (8%) of2-benzyl-5-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a white solid.

LC-MS: (ES, m/z): 333 [M+H]⁺

¹H NMR (300 MHz, DMSO, ppm) 13.36 (s, 1H), 7.86 (d, J=8.7 Hz, 2H),7.38-7.21 (m, 5H), 7.09 (d, J=9 Hz, 2H), 6.26 (s, 1H), 4.10 (s, 2H),3.83 (s, 3H)

EXAMPLE 202-(4-Fluorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1.5-(1-Acetyl-1H-indazol-5-yl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(4-fluorophenyl)-1H-1,2,4-triazol-5-amine (120 mg,0.67 mmol) in diphenyl ether (2 ml) was added ethyl3-(1-acetyl-1H-indazol-5-yl)-3-oxopropanoate (650 mg, 2.36 mmol) andTsOH (5.76 mg, 0.03 mmol), and the resulting mixture was stirred for 5 hat 170° C. The reaction was then quenched by the addition of ethyl ether(8 ml). The solids were collected by filtration to afford5-(1-acetyl-1H-indazol-5-yl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a light yellow crude solid (100 mg, crude).

LC/MS (ES, m/z): [M+H]⁺ 389.0

Step 2.2-(4-Fluorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of5-(1-acetyl-1H-indazol-5-yl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one(100 mg, crude) in methanol (10 ml) was added a solution of potassiumcarbonate (50 mg, 0.36 mmol), and the resulting mixture was stirredovernight at room temperature. The solids were collected by filtrationand washed with water (3 ml) and methanol (10 ml) to afford2-(4-fluorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a light yellow solid (55 mg, 23% 2 steps).

LC/MS (ES, m/z): [M+H]⁺ 347.0

¹H NMR (300 MHz, DMSO) δ 13.12 (s, 1H), 8.43 (s, 1H), 8.16-8.21 (m, 3H),8.07-8.10 (m, 1H), 7.56 (d, J=8.7 Hz, 1H), 7.29-7.35 (t, J=8.7 Hz, 2H),6.20 (s, 1H)

EXAMPLE 215-(1-Ethyl-1H-indazol-5-yl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-(4-fluorophenyl)-1H-1,2,4-triazol-5-amine (100 mg,0.56 mmol) in diphenyl ether (1 ml) was added ethyl3-(1-ethyl-1H-indazol-5-yl)-3-oxopropanoate (400 mg, 1.540 mmol) andTsOH (6 mg, 0.03 mmol), and the resulting mixture was strirred for 2.5 hat 170° C. The reaction mixture was then quenched by the addition ofethyl ether (8 ml). The solids were collected by filtration and washedwith dichloromethane (5 ml) to afford5-(1-ethyl-1H-indazol-5-yl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a light yellow solid (69 mg, 24%).

LC/MS (ES, m/z): [M+H]⁺ 375.0

¹H NMR (300 MHz, DMSO) δ 13.60 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H),8.17-8.21 (m, 2H), 7.88 (d, J=6.0 Hz, 2H), 7.38-7.44 (t, J=8.7 Hz, 2H),6.39 (s, 1H), 4.48-4.55 (m, 2H), 1.40-1.45 (t, J=7.2 Hz, 3H)

EXAMPLE 225-(1H-Benzo[d][1,2,3]triazol-6-yl)-2-(2-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 2-Chlorobenzoyl Chloride

A mixture of 2-chlorobenzoic acid (10 g, 63.87 mmol, 1.00 equiv) andthionyl chloride (10 mL) was stirred overnight at 100° C. After thereaction was completed, the resulting mixture was concentrated undervacuum. This resulted in 10 g (100%) of 2-chlorobenzoyl chloride asyellow oil.

Step 2. 2-(2-Chlorobenzoyl)hydrazinecarboximidamide

A mixture of 2-chlorobenzoyl chloride (10 g, 60 mmol, 1.00 equiv) andhydrazinecarboximidamide carbonic acid salt (11.6 g, 85 mmol, 1.49equiv) in pyridine (10 mL) and dichloromethane (100 g, 1.18 mol, 41.21equiv) was stirred for 3 h at 25° C. After the reaction was completed,the resulting mixture was concentrated under vacuum. The residue waspurified via silica gel column (ethyl acetate/petroleum ether (10/1))resulting in 3 g (22%) of 2-(2-chlorobenzoyl)hydrazinecarboximidamide asyellow oil.

LC-MS (ES, m/z): [M+H]⁺ 213

Step 3. 3-(2-Chlorophenyl)-1H-1,2,4-triazol-5-amine

A solution of 2-(2-chlorobenzoyl)hydrazinecarboximidamide (3 g, 14.1mmol) in water (10 mL) was stirred for 4 h at 100° C. After the reactionwas completed, the solids were collected by filtration, and washed withH₂O (3×5 ml). This resulted in 1.5 g (55%) of3-(2-chlorophenyl)-1H-1,2,4-triazol-5-amine as a yellow oil.

LC-MS: (ES, m/z): [M+H]⁺ 195

¹H NMR (300 MHz, DMSO): 12.44 (brs, 1H), 7.77-7.82 (m, 1H), 7.35-7.57(m, 3H), 6.06 (s, 2H)

Step 4.5-(1H-Benzo[d][1,2,3]triazol-6-yl)-2-(2-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

A mixture of 3-(2-chlorophenyl)-1H-1,2,4-triazol-5-amine (50 mg, 0.26mmol, 1.00 equiv), ethyl 3-(1H-1,2,3-benzotriazol-5-yl)-3-oxopropanoate(100 mg, 0.43 mmol, 1.67 equiv), butan-1-ol (1 mL), and p-TsOH (5 mg,0.03 mmol, 0.11 equiv) was stirred for 1 h at 170° C. in an oil bath.After the reaction was completed, the solids were collected byfiltration and washed with MeOH (3×1 ml). This resulted in 25.3 mg (27%)of5-(1H-benzo[d][1,2,3]triazol-6-yl)-2-(2-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a grey solid.

LC-MS (ES, m/z): [M+H]⁺ 364

¹H NMR (300 MHz, CD₃OD): 8.55 (d, J=5.1 Hz, 1H), 8.15-8.47 (m, 1H),7.92-7.98 (m, 1H), 7.82-7.87 (m, 1H), 7.41-7.50 (m, 3H), 6.54 (s, 1H)

EXAMPLE 235-(1H-benzo[d][1,2,3]triazol-6-yl)-2-o-tolyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

Step 1. 2-Methylbenzoyl Chloride

A solution of 2-methylbenzoic acid (10 g, 1.00 equiv) in thionylchloride (50 mL) was refluxed overnight at 80° C. in an oil bath. Thereaction mixture was concentrated under vacuum to afford 2-methylbenzoylchloride (crude) as a colorless oil.

Step 2. 2-(2-Methylbenzoyl)hydrazinecarboximidamide

To a solution of hydrazinecarboximidamide carbonic acid salt (12.0 g,1.2 eq) in pyridine (100 mL) was added 2-methylbenzoyl chloride (crudeafforded in step 1, 1.0 eq) dropwise at 0° C. The mixture was stirredovernight at room temperature. The reaction mixture was concentratedunder vacuum, diluted with water (30 mL), adjusted to pH 12 with NaOH(aq), and the solid was collected by filtration and washed with water(20 mL) to afford 2-(2-methylbenzoyl)hydrazinecarboximidamide (5.0 g,35% of two steps) as a white solid.

LCMS (ES, m/z): [M+H]⁺ 193.0

¹H-NMR: (DMSO, ppm): 10.11 (brs, 1H), 7.06-7.52 (m, 6H), 6.19 (s, 2H),4.67 (s, 1H), 2.42 (s, 3H), 2.40 (s, 2H)

Step 3. 3-o-Tolyl-1H-1,2,4-triazol-5-amine

A solution of 2-(2-methylbenzoyl)hydrazinecarboximidamide (5 g) in water(40 mL) was stirred overnight at 100° C. The reaction mixture wasdiluted with H₂O (20 mL) and the solid was collected by filtration andwashed with water (20 mL) to afford 3-o-tolyl-1H-1,2,4-triazol-5-amine(3 g, 66% yield) as a white solid.

LCMS (ES, m/z): [M+H]⁺ 175.0

¹H-NMR: (DMSO, ppm): 12.10 (s, 1H), 7.81 (s, 1H), 7.20 (s, 3H), 6.00 (s,2H) 2.54 (s, 3H)

Step 4.5-(1H-Benzo[d][1,2,3]triazol-6-yl)-2-o-tolyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

To a solution of 3-o-tolyl-1H-1,2,4-triazol-5-amine (150 mg, 1.0 eq) inn-BuOH (2 ml) was added ethyl3-(1H-benzo[d][1,2,3]triazol-5-yl)-3-oxopropanoate (240 mg, 1.2 eq) andTsOH (20 mg) and the reaction was stirred for 5 h at 120° C. in an oilbath. The product was collected by filtration and washed with MeOH (3×1ml) to afford5-(1H-benzo[d][1,2,3]triazol-6-yl)-2-o-tolyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one(117.6 mg, 40% yield) as a yellow solid.

LCMS (ES, m/z): [M+H]⁺ 344.0

¹H-NMR: (DMSO, ppm): 16.05 (s, 1H), 13.80 (s, 1H), 12.23 (brs, 1H), 8.65(s, 1H), 7.99-8.01 (m, 3H), 7.37-7.43 (m, 3H), 6.50 (s, 1H), 2.68 (s,1H)

EXAMPLE 245-(1H-benzo[d][1,2,3]triazol-6-yl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide

Step 1. Methyl5-(1H-benzo[d][1,2,3]triazol-6-yl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate

To a solution of ethyl3-(1H-benzo[d][1,2,3]triazol-6-yl)-3-oxopropanoate (246 mg, 1.5 eq) inn-BuOH (5 ml) was added TsOH (10 mg, 0.1 eq) and methyl5-amino-1H-1,2,4-triazole-3-carboxylate (100 mg, 1.0 eq), and thereaction mixture was stirred for 20 h at 130° C. The solids werecollected by filtration and washed with methanol (10 ml) to affordmethyl5-(1H-benzo[d][1,2,3]triazol-6-yl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate(80 mg, 60%).

LCMS (ES, m/z): [M+H]⁺ 312.0

Step 2.5-(1H-Benzo[d][1,2,3]triazol-6-yl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide

Methyl5-(1H-benzo[d][1,2,3]triazol-6-yl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate(80 mg, 1.0 eq) in methanolic ammonia (8 ml) was stirred overnight at80° C. Then the pH value of the solution was adjusted to pH 4 with HCL(3N). The solids were collected by filtration, washed with water (10 ml)and purified by prep-HPLC to afford5-(1H-benzo[d][1,2,3]triazol-6-yl)-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamideas a white solid (34.8 mg, 45%). LCMS (ES, m/z): [M+H]⁺ 297.0

¹H NMR (300 MHz, DMSO) δ 8.52 (s, 1H), 8.15 (d, J=7.2 Hz, 2H), 7.96 (s,1H), 7.86 (s, 1H), 7.54 (s, 1H), 7.19 (s, 2H), 6.33 (s, 1H), 4.48-4.55(m, 2H), 1.39-1.44 (t, J=7.2 Hz, 3H)

EXAMPLE 25

To a solution of 3-(2-chlorophenyl)-1H-1,2,4-triazol-5-amine (50 mg,0.25 mmol) in n-BuOH (0.2 mL) was added ethyl3-(1-ethyl-1H-indazol-5-yl)-3-oxopropanoate (100 mg, 0.37 mmol) andp-TsOH (10 mg) at room temperature. After refluxing overnight, thesolids were collected by filtration and washed with methanol (3×2 mL) toafford2-(2-chlorophenyl)-5-(1-ethyl-1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-oneas a yellow solid (39.5 mg, 40%).

LCMS (ES, m/z): [M+H]⁺ 391.1

¹H-NMR (300 MHz, DMSO) δ 13.66 (s, 1H), 8.37 (s, 1H), 8.25 (s, 1H),7.98-7.95 (m, 1H), 7.86 (s, 2H), 7.67-7.61 (m, 1H), 7.58-7.46 (m, 2H),6.41 (s, 1H), 4.51 (q, J=7.2 Hz, 2H), 1.42 (t, J=7.2 Hz, 3H)

EXAMPLE 26 5-(4-Methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 275-(4-Methoxyphenyl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 285-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 295-(Benzo[d][1,3]dioxol-5-yl)-2-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 305-(2-Methoxyphenyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 31 2,5-Diphenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 325-(4-Methoxyphenyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 335-(3-Methoxyphenyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 342-(4-Chlorophenyl)-5-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 352-(4-Methoxyphenyl)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 362-(2-Chlorophenyl)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 375-(3,4-Dimethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 38 5-(4-Methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 395-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-(4-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 405-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 415-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 422-(4-Chlorophenyl)-5-(1-ethyl-1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 435-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 442-(4-Chlorophenyl)-5-(1H-indazol-5-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

EXAMPLE 455-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-benzyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one

The activity of the compounds in Examples 1-45 as PASK modulators isillustrated in the following assays.

Biochemical Assay for hPASK Activity PASK ATP Radiochemical Assay

Purified PASK (UniProt #Q96RG2; human recombinant N-terminal GST taggedconstruct, residues 879-1323) from insect cells (final concentration 5nM) is added to freshly prepared Base Reaction Buffer containing 20 mMHEPES (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA,0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO and Myelin Basic Protein (20 μM final).Test compounds in DMSO are then added and the mixture, followed bydelivery of ³³P-ATP (specific activity 0.01 μCi/μl final) to initiatethe reaction. The kinase reaction is incubated for 120 min at roomtemperature. The entire reaction mixture is washed through onto a P81Phosphocellulose paper and washed three times for 10 minutes in 75 mMphosphoric acid and once in methanol prior to drying and scintillationcounting.

Results for this assay are shown below in Table 1. Examples not listedin the table were not tested.

TABLE 1 IC₅₀ Kinase Domain + indicates ≦10 um Example # − indicates >10um 8 + 9 + 15 + 30 + 31 + 32 + 33 + 34 + 35 + 36 + 37 + 38 +

PAS Kinase FRET Assay

The aim of the FRET assay is to determine the inhibition potential oftest compounds on targeted kinase. This assay platform provides ahomogenous screening method for measuring kinase activity byquantitating the amount of phospho-substrate in solution following akinase reaction.

In the presence of kinase and ATP, the Ulight-peptide is phosphorylatedand captured by an anti-phospho-substrate antibody, which brings the Euchelate donor and Ulight acceptor dyes into close proximity. Uponexcitation at 340 nm, the Eu chelate transfers its energy to the Ulightdye, resulting in a fluorescent light emission at 665 nm.

Titration of kinase at 1 mM ATP was achieved via the following protocol.After making serial three-fold dilutions of PASK (Invitrogen) inreaction buffer across the plate; 5 μl of kinase dilution and 5 μlsubstrate/ATP mix were added to the wells of the white Optiplate-384(PerkinElmer). The contents of the plate were and incubated at RT for 1h. The reaction was stopped by adding 5 μl of stop solution to each testwell followed by mixing and incubation at RT for 10 minutes. 5 μl ofdetection mix (detection antibody diluted in detection buffer) wasadded; the contents of the plate were mixed and then incubated in thedark for 1 hour at RT. The signal was recorded at TR-FRET mode (665nm/615 nm). The results were graphed to calculate the EC₅₀.

Titration of ATP at the EC₅₀ concentration of kinase to determine ATPKm,app. was performed using the following method. After making serialdilutions of ATP (Invitrogen), 5 μl of ATP dilution and 5 μlsubstrate/kinase mix were added to the wells of the white Optiplate-384(PerkinElmer). The contents of the plate were and incubated at RT for 1h. The reaction was stopped by adding 5 μl of stop solution to each testwell followed by mixing and incubation at RT for 10 minutes. 5 μl ofdetection mix (detection antibody diluted in detection buffer) wasadded; the contents of the plate were mixed and then incubated in thedark for 1 hour at RT. The signal was recorded at TR-FRET mode (665nm/615 nm). The results were graphed to calculate the EC₅₀ as the ATPKm,app.

Compound screening was done via the following method. 10 mM stocksolution of test compound in DMSO was prepared by dissolving testcompound in DMSO at RT for 1 hour, and then sonicating at 100% outputfor 8 minutes. If compound is not soluble under this condition, it wasdiluted to 3 mM. Kinase reaction buffer was prepared containing 10 mMMgCl₂, 50 mM HEPES, 1 mM EGTA, 0.01% TWEEN-20, 2 mM DTT. Serialdilutions of the test compounds were prepared at 4×final assayconcentrations using Freedom EVO200® dispensing system as follows:12×10⁻⁵ M, 4×10⁻⁵ M, 1.33×10⁻⁵ M, 4.44×10⁻⁶ M, 1.48×10⁻⁶ M, 4.92×10⁻⁷ M,1.65×10⁻⁷ M, 5.48×10⁻⁷ M, 1.82×10⁻⁸ M, 6.09×10⁻⁹, 2.03×10⁻⁹ M. Testcompounds (2.5 μl at 4×the final assay concentration) was added to wellsusing Freedom EVO200® dispensing system. As a positive control, 2.5 μlof positive compound was added to assay wells, and 2.5 μl of DMSO toassay wells as vehicle control. Kinase solution was prepared in reactionbuffer at 2×final assay concentration. Kinase solution (5 μl) was addedto each well of the assay plate. The substrate and ATP solution wasprepared in kinase reaction buffer at 4×final assay concentration. Thekinase reaction was started by adding 2.5 μl of substrate+ATP mixsolution to each well of the assay plate. The plate is mixed on a plateshaker; then covered and allowed to react for 2 hours in the dark at 25°C. without shaking. The reaction was stopped by adding 5 μl of stopsolution to each test well followed by mixing and incubation at RT for10 minutes in the dark. 5 μl of detection mix (detection antibodydiluted in detection buffer) was added; the contents of the plate weremixed and then incubated in the dark for 1 hour at RT. The signal wasrecorded at TR-FRET mode (665 nm/615 nm).

Results are shown below in Table 2.

TABLE 2 IC₅₀ Kinase Domain + indicates ≦10 um Example # − indicates >10um 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + 15 +16 + 17 + 18 + 19 + 20 + 21 + 22 + 23 + 24 + 25 + 26 + 27 + 28 + 29 +34 + 39 + 40 + 41 + 42 + 43 + 44 + 45 +

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A compound of structural Formula I

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein: R₁ is chosen from hydrogen, aryl, alkyl, arylalkyl, heteroaryl,heteroarylalkyl, and CONR₁₀R₁₁, any of which may be optionallysubstituted. R₂ is chosen from aryl and heteroaryl, either of which maybe optionally substituted; and R₁₀ and R₁₁ are each independently chosenfrom hydrogen, lower alkyl, aryl, or optionally, R₁₀ and R₁₁ can betaken together to form a heterocycloalkyl, or heteroaryl.
 2. Apharmaceutical composition comprising a compound as recited in claim 1together with a pharmaceutically acceptable carrier.
 3. A compound asrecited in claim 1 for use as a medicament.
 4. A compound as recited inclaim 1 for use in the manufacture of a medicament for the prevention ortreatment of a disease or condition ameliorated by the inhibition ofPASK.
 5. A compound as recited in claim 1 for use in the prevention ortreatment of a disease or condition ameliorated by the inhibition ofPASK.
 6. A pharmaceutical composition comprising a compound as recitedin claim 1 together with a pharmaceutically acceptable carrier.
 7. Amethod of inhibiting PASK comprising contacting PASK with a compound asrecited in claim
 1. 8. A method of treatment of a disease comprising theadministration of a therapeutically effective amount of a compound asrecited in claim 1 to a patient in need thereof.
 9. The method asrecited in claim 8 wherein said disease is chosen from cancer and ametabolic disease.
 10. The method as recited in claim 9 wherein saiddisease is a metabolic disease.
 11. The method as recited in claim 10wherein said metabolic disease is chosen from metabolic syndrome,diabetes, dyslipidemia, fatty liver disease, non-alcoholicsteatohepatitis, obesity, and insulin resistance.
 12. The method ofclaim 11 wherein said diabetes is Type II diabetes.
 13. The method ofclaim 11 wherein said dyslipidemia is hyperlipidemia.
 14. The method ofclaim 13 wherein said hyperlipidemia is hypertriglyceridemia.
 15. Amethod for achieving an effect in a patient comprising theadministration of a therapeutically effective amount of a compound asrecited in claim 1 to a patient, wherein the effect is selected from thegroup consisting of reduction of triglycerides, reduction ofcholesterol, and reduction of hemoglobin A1c.
 16. The method of claim 15wherein said cholesterol is chosen from LDL and VLDL cholesterol. 17.The method of claim 15 wherein said triglycerides are chosen from plasmatriglycerides and liver triglycerides.
 18. A method of treatment of aPASK-mediated disease comprising the administration of: a. atherapeutically effective amount of a compound as recited in claim 1;and b. another therapeutic agent.